Spiders are capable of producing a variety of silk types, each with their own unique protein composition and function. Dragline silk in particular, has been of great interest due to its high tensile strength and extensibility. In the past, synthetically produced dragline fibers have not been able to match the superior properties of natural silk. A recent discovery in the western black widow spider, Latrodectus hesperus, might be the missing link between the current state of synthetic silks and naturally produced fibers. Our research is centered around the discovery a new family of five low-molecular-weight cysteine-rich proteins (CRPs) and their potential function within dragline silk. This study focuses on the characterization of recombinantly expressed CRP1, CRP2, and CRP4. Through structural analysis using circular dichroism, it has been determined that the CRP family members have mostly alpha-helical secondary structure and exhibit small differences in their ability to maintain their structure in the presence of changing environmental conditions. The study also covers the effects of temperature and pH on the folding and unfolding of the CRPs. It appears that pH is the dominant influence on protein unfolding within the major ampullate gland.

Spider silk is a material that both has impressive mechanical properties and is also environmentally friendly. Though there are limitless potential engineering applications for such materials, industrial production of spider silk has proven to be challenging. Farming silk from spiders, as is done with silkworms, is not a viable option for large-scale production of spider silk due to the venomous and predatory nature of spiders. Here, an attempt is made to express synthetic spider silk minifibroins heterologously in Escherichia coli, to purify the recombinant spidroins from cell lysate, and to spin them into artificial fibers through a biomimetic process. Silk minifibroins were designed to be similar to Major Ampullate Spidroin 1 from Latrodectus hesperus. Synthetic fibers were examined by scanning electron and light microscopy, and their mechanical properties were tested by a tensometer. Properties of synthetic silk were compared to those of native dragline silk from the same species from which their design was inspired, revealing synthetic silk fibers with lower breaking stress and breaking strain.

To mitigate climate change a proposed space-based geoengineering solutionis to screen off solar irradiance by placing a membrane in between the Earthand the Sun. The feasibility of such a project largely depends on minimizingthe mass of the shading screen and as an extension to the Sunshade projectthis thesis investigated how such a low-mass membrane could be designed.Because of the acting forces at location in space, minimizing the mass impliesthat the material ought to have a low reflection coefficient and surface densityand therefore the highly transparent material of artificial spider silk was chosenas the proposed material. The only possibility to block light is then byrefraction or diffraction and, since the presence of apertures might lower thesurface density, the structure of the suggested membrane is a grid patternof wires. Such a diffraction grating was investigated while applying twomethods. Method 1 optimized the dimensions of the structure to lower thetotal transmission on Earth when placed on the direct transmission axis ofthe membrane and method 2 tilted the membrane in order to place Earth ata diffraction minimum. This resulted in three suggested designs A, B, andC with surface densities varying from that of 0.00867 to 0.228 gm−2. Theresults were compared with two previous design proposals where the lowestareal density was 0.34g/m2, which is 3/2 to 40 times larger than the densitiesproposed in this paper. The reflectivities for A and B were 12.5 and 3.75 timeslarger than that of the smallest previously achieved reflectivity. The reflectivityof C could not be determined exactly but ought to have a reflectivity at leastas low as B at 3%, making it the most promising candidate for a membranedesign of the three.

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Sticky prey capture threads are produced by many members of the spider Infraorder Araneomorphae. Cribellar threads are plesiomorphic for this clade, and adhesive threads are apomorphic. The surface of cribellar thread is formed of thousands of fine fibrils. Basal araneomorphs produce cylindrical fibrils, whereas more derived members produce fibrils with nodes. Cribellar fibrils snag and hold rough surfaces, but other forces are required to explain their adherence to smooth surfaces. Threads of Hypochilus pococki (Hypochilidae) that are formed of non-noded fibrils hold to a smooth acetate surface with the same force under low and high humidities. In contrast, threads of Hyptiotes cavatus and Uloborus glomosus (Uloboridae) that are formed of noded fibrils hold with greater forces to the same surface at intermediate and high humidities. Threads spun by eight species representing seven genera and four families with noded fibrils absorb water, while that of two families, represented by one species each with smooth fibrils, repel water, indicating increase hygroscopisity associated with the presence of nodes. Additionally, equations describing van der Waals and hygroscopic forces can predict the observed stickiness of these threads. This model supports the hypothesis that van der Waals forces allow non-noded cribellar fibrils to adhere to smooth surfaces, whereas noded fibrils employ van der Waals forces at low humidities and add hygroscopic forces at higher humidities. Thus, there appear to have been two major events in the evolution of spider prey capture thread: the addition of hydrophilic nodes to the fibrils of cribellar threads and the replacement of cribellar fibrils by glycoprotein glue. / Master of Science

Spider silk is a material that both has impressive mechanical properties and is also environmentally friendly. Though there are limitless potential engineering applications for such materials, industrial production of spider silk has proven to be challenging. Farming silk from spiders, as is done with silkworms, is not a viable option for large-scale production of spider silk due to the venomous and predatory nature of spiders. Here, an attempt is made to express synthetic spider silk minifibroins heterologously in Escherichia coli, to purify the recombinant spidroins from cell lysate, and to spin them into artificial fibers through a biomimetic process. Silk minifibroins were designed to be similar to Major Ampullate Spidroin 1 from Latrodectus hesperus. Synthetic fibers were examined by scanning electron and light microscopy, and their mechanical properties were tested by a tensometer. Properties of synthetic silk were compared to those of native dragline silk from the same species from which their design was inspired, revealing synthetic silk fibers with lower breaking stress and breaking strain.

Immobilizing antimicrobial substances onto biocompatible materials is an important approach for the design of novel, functionalized medical devices. By choosing antimicrobial substances from innate immune systems, the risk for development of resistance in pathogenic microbes is lower than if conventional antibiotics are used. Combining natural antimicrobial peptides and bactericidal enzymes with strong and elastic spider silk through recombinant protein technology would enable large-scale production of materials that could serve as functionalized wound dressings. Herein, fusion proteins with the engineered spider silk sequence 4RepCT and five different antimicrobial substances were constructed using two different strategies. In the first, the fusion proteins had a His-tag as well as a solubility-enhancing domain N-terminally to the antimicrobial agent during expression. The tags were cleaved and separated from the target protein during the purification process. The other approach provided a His-tag but no additional solubility domain. The antimicrobial agents included in the work were a charge engineered enzyme and four antimicrobial peptides herein called Peptide A, Peptide B, Peptide C and Peptide D. Four out of five fusion proteins could be expressed in Escherichia coli without exhibiting noticeable toxicity to the host. However, most target proteins were found in the non-soluble fraction. For D-4RepCT, neither soluble nor non-soluble proteins were identified. An operating strategy for expression and purification of antimicrobial spider silk proteins was developed, where the construct system providing the solubility-enhancing domain N-terminally to the antimicrobial sequence, and long time expression at low temperatures is a promising approach. The fusion proteins A-4RepCT and C-4RepCT could be produced in adequate amounts, and they proved to possess the ability to assemble into stable fibers. When incubating solutions of Escherichia coli on the functionalized silk material A-4RepCT, it showed to decrease the number of living bacteria in solution, in contrary to wild-type 4RepCT on which bacteria continued to proliferate. Initial studies of the viability of bacteria adhered to the surface of the functionalized spider silk are so far inconclusive. A larger sample size, complementary experiments and methodology optimization is needed for a proper assessment of antibacterial properties. However, preliminary results for the development of antimicrobial spider silk are positive, and the approach elaborated in this work is believed to be applicable for the construction of functional spider silk with a wide range of natural antimicrobial agents for future wound healing applications.

Spiders produce a diverse number of silk proteins that are well-known for their extraordinary mechanical and biological properties. Dragline silk has been the most prominent focus of research because of its exceptional high tensile strength and extensibility. In our research, we have focused on the characterization of small molecular weight proteins found within dragline and tubuliform silks. Within the black widow spider, Lactrodectus hesperus, these proteins have been named Cysteine-Rich Protein (CRP) and determined to be a family of five individual proteins. The small protein identified within the tubuliform silks has been named Egg Case Protein 3 (ECP-3). In this study, recombinant expression of ECP-3 in the pET-19b-SUMO vector was to facilitate purification and development of an immunological reagent. Using western blot analysis, we have demonstrated that ECP-3 is efficiently expressed in bacteria. We also investigated CRP1 protein and its ability to bind MaSp1 components using pull down assays to determine potential interactions. No substantial biochemical evidence was produced to demonstrate protein-protein interactions between the two. Additionally, we show that using RT-PCR analysis from mRNAs collected from the major ampullate gland that transcript levels for CRP-family members from non-silked and a silked spider are different. CRP2 and CRP4 mRNA levels were shown to increase upon silking. Overall, the major findings of this thesis involved characterizing the ECP-3 protein found within tubuliform silks as well as determining the expression patterns for CRP-family members.

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In this study we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus , whose DNA coding sequences have not been reported. Based upon solubility differences in 8 M guanidine hydrochloride, it is demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining that the egg case silk is relatively complex at the molecular level, containing a large number of proteins with differing molecular weights. Protein components of egg case silk with a size about 100 kDa were obtained by a solubilization time course study, which indicates these proteins are likely to be embedded in the silk filament. Peptides in these 100 kDa proteins were released by tryptic in-gel and in-solution digestion. The peptides were sequenced using a MALDI tandem TOF mass spectrometer. Some of the de novo sequences were confirmed using a linear ion trap mass spectrometer equipped with a nanospray ion source. Combining the peptide sequences obtained, reverse genetics was employed to trace silk genes encoding proteins containing these de novo peptides. Three silk protein coding sequences were successfully discovered, which encode silk proteins named 3B, T1 and ECSP-1, respectively. 3B and T1 show the standard fibroin protein pattern. Amino acid repeat patterns were observed in these two silk clones. But the amino acid compositions of 3B and T1 show differences with the total amino acid composition of egg case silk, and also, the peptide sequences cannot be found in the primary amino acid sequences of 3B and T1. ECSP-1 protein represents one of the egg case silk proteins with a size of about 100 kDa. A number of peptide sequences obtained by mass spectrometric de novo sequencing were successfully located in ECSP-1's primary amino acid sequence. Sequence analysis demonstrates ECSP-1 represents a new class of silk proteins, with fibroin-like properties. The expression pattern of ecsp-1 is largely restricted to the tubuliform gland inside of the L. hesperus spider, with lower levels detected in the major and minor ampullate glands, which also confirms the identity of ECSP-1. It is also demonstrated that ECSP-1 assembles into higher aggregate structures through the formation of disulfide bonds. Peptide sequences from silk proteins from the Tarantula spider Grammostola rosea were also obtained. These sequences will be beneficial in obtaining genes encoding the silk from this spider species.